Pro-melanocortins are present in high levels in the pituitary and are processed into three major peptide hormone fragments: adrenocorticotrophin (ACTH), the melanocyte stimulating hormones, and β-endorphin. The various melanocyte stimulating hormones are known to activate the MC4 (melanocortin subtype 4) receptor. The MC4 receptor has been linked to the regulation of appetite (1). Evidence has suggested that melanocortins are involved in mediating appetite regulation through the effects of leptin and controlling the expression of neuropeptide Y. Thus, the MC4 receptor is a potential target for anti-obesity agents (2).

This application note describes a 384-well melanocortin MC4 receptor binding assay, which has been developed on the LEADseeker™ Multimodality Imaging System.

* Use of the receptor in this application note is not permitted in North America. For further information on the use of this receptor in restricted territories, please contact Euroscreen’s Business Development and Licensing Manager at vlannoy@euroscreen.be

6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min with quasi-coincident averaging and 3 ×3 binning.

7. For background estimation, bead-only wells (31.25 µg of bead/well, 20 µl), plus assay buffer (20 µl) were included in all experiments. Typically backgrounds exhibited from bead-only wells were less than 10 integrated optical density (IOD) units, and were routinely subtracted from total and NSB values during data analyses.

ResultsKd determination
Saturation binding was carried out with dilutions of [125I] NDP-α-MSH to give a range of concentrations from 4.05 pM to 10.43 nM in the assay wells. Figure 1 shows the saturation curve which was fitted using non-linear regression with the data analysis package GraphPad Prism v4.0. A Kd value of 0.931 nM (95% confidence intervals 0.744 to 1.117 nM) was estimated directly from the curve
.

DMSO toleranceDilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.5 to 32% (v/v) in the assay wells. From the results shown in Figure 2, it can be seen that the assay was tolerant of DMSO up to a final concentration 2% (v/v).

Time courseA time course experiment was carried out using reagent concentrations as described in the protocol. Measurements were made over an assay incubation time of 20.5 h. The assay reached equilibrium after 8-h incubation at room temperature and the signal was stable for at least another 12.5 h (Fig 4).

Z’ analysisA Z’ analysis was carried out using 48 replicate values for total and NSB wells. Z’ was 0.796 (Fig 5), which confirmed the robustness of the assay (3).

Conclusion
The melanocortin MC4 receptor binding assay has been successfully miniatur
ized to a 384-well format on LEADseeker Multimodality Imaging System and is suitable for high-throughput screening formats. The assay is tolerant up to 2% DMSO (v/v); is robust, exhibiting a Z’ value of 0.789, and has a stable signal of at least 12.5 h.

(Date:10/12/2017)... ... October 12, 2017 , ... The Blavatnik Family Foundation ... six Finalists of the 2017 Blavatnik Regional Awards for Young Scientists. Established ... administered by the New York Academy of Sciences to honor the excellence of ...

(Date:10/12/2017)... ... October 12, 2017 , ... ... launched Rosalind™, the first-ever genomics analysis platform specifically designed for life science ... in honor of pioneering researcher Rosalind Franklin, who made a major contribution ...

(Date:10/11/2017)... ... October 11, 2017 , ... ... today it will be hosting a Webinar titled, “Pathology is going digital. Is ... , on digital pathology adoption best practices and how Proscia improves lab economics ...

(Date:10/11/2017)... Tampa Bay, Florida (PRWEB) , ... October 11, ... ... Food and Drug Administration (FDA) has granted orphan drug designation to SBT-100, its ... antibody (sdAb) for the treatment of osteosarcoma. SBT-100 is able to cross the ...

(Date:6/14/2017)... 15, 2017 IBM (NYSE: IBM ) is introducing several ... dedicated to developing collaboration between startups and global businesses, taking ... During the event, nine startups will showcase the solutions they ... industries. France is ... with a 30 percent increase in the number of startups ...